Gene therapy is emerging as a popular form of treatment that aims to address a variety of disease states through the transfer of functional genetic material into cells. Conventionally, gene therapy is carried out by including a transgene composed of a cDNA or, less commonly, the genomic configuration of the transgene (including the exons and introns) into a gene delivery vehicle (e.g., a gene transfer vector). Typically, there is a limit to the amount of DNA or RNA (i.e., transgene) that can be packaged in a gene therapy vector. To minimize the size occupied by the transgene, expression cassettes containing the transgene generally contain cDNA rather than genomic clones. However, in some cases, gene therapy vectors have been constructed to contain introns. For example, a gene therapy vector useful in treating cystic fibrosis has been described which includes an intron-containing region in the 5′ end of the primary transcript of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The intron-containing region in this vector is a hybrid between the cytomegalovirus (CMV) immediate early intron and an intron from the human immunoglobulin gene. The rationale for using an intron at the 5′ end of the primary transcript is based on two considerations. First, in vitro transfection studies indicate that such genes introduced into cells are expressed more efficiently when the primary transcripts contain introns. Second, an intron at the 5′ end of the primary transcript is convenient for PCR analyses to distinguish vector DNA from the mRNA transcript. Genomic clones have rarely been used in gene therapy with the exception of relatively small genomes (e.g., erythropoietin) or in the gutless adenovirus vectors for which a genomic clone of the human α1-antitrypsin gene has been used as a transgene. In vivo data using the gutless vector have shown prolonged expression of the transgene, but this observation was interpreted to reflect the properties of the gutless vector rather than the nature of the transgene. In contrast to cDNA, pre-mRNA splicing of genomic DNA is essential for protein production.
RNA splicing is part of a process whereby primary transcripts made by transcription of a DNA template by an RNA polymerase are rearranged to make messenger RNA (mRNA). For most mammalian genes, one or more introns present in the primary transcript are removed, leaving only the exons which, when spliced together, constitute the mature mRNA. The number and length of introns differ greatly among genes.
RNA splicing is mediated by the spliceosome, which is a large protein/RNA complex responsible for removing introns in a two-step process. First, cleavage occurs at the splice donor site (5′ end of intron) exposing a 5′-phosphate, which immediately ligates to a 2′-OH at the branch point that is located close to the splice acceptor within the intron. Secondly, there is a cleavage at the splice acceptor site with the simultaneous ligation of the 3′-OH of the upstream exon to the 5′-phosphate of the downstream exon. These two steps result in the release of the intron as a lariat with a free 3′-OH end.
In the process of constitutive splicing, all of the introns are excised from the pre-mRNA to give a unique mRNA species. However, a number of genes have been described for which more than one mRNA species is derived from a single pre-mRNA. Such an occurrence is referred to as alternative splicing. When this takes place, many different isoforms of a protein can be produced from a pre-mRNA. Moreover, each of these isoforms can have different biological activities. An example of an alternatively spliced pre-mRNA transcript is demonstrated by the angiogenic factor VEGF. The VEGF-A (sometimes referred to as “VEGF-1”) gene contains 8 exons and 7 introns that, by alternative splicing, can form at least six isoforms of the protein.
The longest protein isoform is VEGF206, whose mRNA contains the entirety of all eight exons encoding a pre-protein of 232 amino acids, which is processed to the mature form of 206 amino acids. Alternative splicing to produce the different isoforms is focused around exons 6, 7, and 8. The VEGF121 isoform results from joining the splice donor at the end of exon 5 directly to the splice acceptor in exon 8, thereby completely eliminating exons 6 and 7. Exon 6 is especially complex with three different potential splice donors which can ligate to exon 7, resulting in the VEGF206, VEGF189, and VEGF183 isoforms. The 3′ non-translated end of the gene contains regulatory elements that increase mRNA half-life in response to ischemia. Since the mRNAs for all isoforms share the same 5′ and 3′ end, RT-PCR can be used to estimate the relative amounts in any tissue. In most normal tissues, VEGF165 and VEGF189 are most abundant, with changes in expression related to neoplastic transformation (see, e.g., Jackson et al., J. Urol., 157, 2323-2328 (1997), and Cheung et al., Hum. Pathol., 29, 910-914 (1998)). For example, in carcinomas originating in lung or colon, a switch to the shorter VEGF121 isoform was observed (see, e.g., Cheung et al. (1998), supra). In non-small cell lung cancer, an increase in VEGF189 has been associated with a more aggressive form of the disease, indicating a poor prognosis for the patient (see, e.g., Tokenaga et al., Br. J. Cancer., 77, 998-1002 (1998)). In addition, the VEGF145 isoform was initially discovered in carcinomas (see, e.g., Poltorak et al., J. Biol. Chem., 272, 7151-7158 (1997)) but has not been observed in non-transformed tissues (see, e.g., Jackson et al. (1997), supra, and Cheung et al. (1998), supra).
The significance of the VEGF isoforms is in their different biological activities. First, the different isoforms have different affinities for the VEGF receptors. At least three VEGF receptors (fltl, flkl/KDR, and neuropilin) are known, which are found in different cell types and at different times during development. Fltl mediates cell migration, while KDR is required for the proliferative effects of VEGF (see, e.g., Barleon et al., Blood, 87, 3336-3343 (1996)). While VEGF165 has approximately equal affinity to the flkl/KDR receptor and the fltl receptor, VEGF 121 has a much lower affinity for fltl and binds primarily to KDR (see, e.g., Keyt et al., J. Biol. Chem, 271, 7788-7795 (1996)). Thus, VEGF121 is expected to be biologically inactive in tissues lacking fltl. In the same way, neuropilin is believed to enhance the interaction of VEGF165 with KDR (but not fltl), but has no effect on the binding of VEGF121 to KDR (see, e.g., Gitay-Goren et al., J. Biol. Chem., 271, 5519-5523 (1996), and Park et al., J. Biol. Chem., 269, 25646-25654 (1994)). Second, the different VEGF isoforms differ in their ability to bind heparin and other negatively charged cell matrix components. VEGF121 is missing the basic domains located in exons 6 and 7 which determine interaction with heparin. The presence of heparin can modify both the affinity of the VEGF for its receptors and the residency time in tissue (see, e.g., Keyt et al. (1996), supra, and Cohen et al. (1995), supra). The heparin binding isoforms, such as VEGF165 and VEGF189, will bind extracellular matrix strongly and can be released as biologically active peptides by proteases such as plasmin (see, e.g., Keyt et al. (1996), supra, Athanassiades et al., Bio. Reprod., 59, 643-654 (1998), and Terman et al., Growth Factors, 11, 187-195 (1994)).
The biological significance of the different properties of VEGF isoforms is proven by the phenotype of mice which are unable to make the heparin binding isoform VEGF164/188 (note that the mice VEGF isoforms are one amino acid shorter than the human homologues) (see, e.g., Carmeliet et al., Nat. Med., 5, 495-502 (1999)). Complete deletion of only the VEGF gene is lethal to a mouse embryo even when only one of the two alleles is deleted (see, e.g., Carmeliet et al., Nature, 380, 435-439 (1996)). However, mice can be made with small genomic deletions which encompass exons 6 and 7, thereby making VEGF120 the only isoform that can be produced (see, e.g., Carmeliet et al. (1999), supra). Homozygote mice for VEGF120 are lethal neonatally and suffer from impaired myocardial angiogenesis, which results in decreased contractility and ischemic cardiomyopathy. Thus, the developmental roles of VEGF can be furnished by VEGF120 while the postnatal development of the blood supply, especially to cardiac muscle, depends on the VEGF164/VEGF188 isoforms. This evidence supports the contention that different therapeutic effects might be expected from the production of different isoforms or mixtures of isoforms of VEGF delivered by gene therapy. It is also conceivable that genes other than VEGF can be used in a similar manner. While alternative splicing can accomplish the production of different isoforms of a particular gene, it would be advantageous to construct nucleic acid molecules that comprise splice sites that promote the production of one isoform of a particular gene over another. Such nucleic acid molecules will allow for more control over splicing and isoform production and will be useful in therapeutic applications, as well as early, sensitive, and accurate methods for measuring the effectiveness of such therapeutic applications in a mammal.
The invention provides such nucleic acid molecules, therapeutic applications, and methods. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.